<DIV style="font-family:Arial, sans-serif; font-size:10pt;"><DIV>Thank you
everyone for your advice, I really appreciate it! I am doing a multiplex
immunofluorescent assay (4 channels) and I am planning to strip the antibodies
and reprobe with a second round of antibodies. The antibodies are
labelled with quantum dots so I am not sure how that will influence things, but
luckily I won't have the DAB problem several of you mentioned. I think I
will try several of these techniques and evaluate which works the best.</DIV>
<DIV> </DIV>
<DIV>Thanks again for all your help,</DIV>
<DIV> </DIV>
<DIV>Claire <BR><BR>--- jkier...@uwo.ca wrote:<BR><BR>From: John Kiernan
<jkier...@uwo.ca><BR>To: histonet@lists.utsouthwestern.edu,
cwes...@valasciences.com<BR>Cc: Amos Brooks
<amosbro...@gmail.com><BR>Subject: Re: [Histonet] Stripping
antibodies<BR>Date: Sun, 30 Oct 2011 12:09:58 -0400<BR><BR></DIV>
<DIV><SPAN>
<DIV>Antibodies are quite easily removed by acidity - pH 1 to 2. This
also removes enzyme-antibody conjugates. The coloured products of the
enzyme histochemical reactions are generally not affected. The brown oxidation
product of DAB (from peroxidase) and the blue products from
bromochloroindoxyl/tetrazolium methods (for alkaline phosphatase) are very
stable. This is good: you can remove all the primary and enzyme-labelled
antibodies without losing track of the stained antigen in the section. A
second immunostaining, with a differently coloured end-product can
follow. If you use AEC as a peroxidase chromogen (red), it should be
for the last immunohistochemical procedure, and you will have to use an
aqueous mounting medium. The AEC oxidation product dissolves in alcohol and
other organic solvents.</DIV>
<DIV> </DIV>
<DIV>Amos suggests a strong oxidation (acid permanganate followed by oxalic
acid to remove brown manganese dioxide) to get rid of the insoluble brown
oxidation product of DAB. Is this what you want? This oxidation
converts the sulphur-containing amino acids, including cystine -S-S-
links, to sulphonic acids, which attract cationic dyes even at pH 1. This
oxidation couuld seriously modify the epitopes for the next applied primary
antibody.</DIV>
<DIV> </DIV>
<DIV>Multicolour immunohistochemistry has been a developed technology for
several years. Any lab needing to do such work needs to take training
courses (hundreds of dollars) or buy a book (tens of dollars). The one by
Chris van der Loos, Immunoenzyme Multiple Staining Methods, Oxford, UK: Bios,
1999, is very good.</DIV>
<DIV> </DIV>
<DIV>John Kiernan</DIV>
<DIV>Anatomy, UWO </DIV>
<DIV>London, Canada </DIV>
<DIV>= = = =</DIV>On 29/10/11, <B>Amos Brooks </B><amosbro...@gmail.com>
wrote:</SPAN>
<BLOCKQUOTE style="BORDER-LEFT: #00f 1px solid; PADDING-LEFT: 13px;
MARGIN-LEFT: 0px">
<DIV>Hi,<BR> Stripping sites are usually found in the more
seedy areas of large<BR>cities ... Oh wait you meant ... nevermind :-) If you
developed the reaction<BR>with DAB, you may be in a difficult position. DAB is
a really strong<BR>reaction. You might try a Mallory bleach to remove it. 0.3%
sulfuric acid in<BR>5% potassium premanganate for 5 min followed by 5% oxalic
acid until it is<BR>clear (usually a couple of minutes). Unfortunately, I am
not sure what<BR>effect this will have on the epitopes you are looking for, but
this is<BR>usually what is used to clean precipitated DAB from instruments it
should<BR>work on the tissue too.<BR> If you use another
chromogen such as AEC it is much easier (to many<BR>people's dismay) to remove
this end product. Using alkaline phosphatase will<BR>be easy to remove the
chromogens as well. It would also be very easy to do<BR>this with fluorescent
tags too. Just remove the coverslip and dip the slide<BR>in ethanol and it will
remove the colored end product. Then just start over<BR>for the new
antigen.<BR><BR>Good luck,<BR>Amos<BR><BR>On Fri, Oct 28, 2011 at 11:50 AM,
<histonet-requ...@lists.utsouthwestern.edu<BR>> wrote:<BR><BR>>
Message: 2<BR>> Date: Thu, 27 Oct 2011 10:30:40 -0700<BR>> From: "Claire
Weston" <cwes...@valasciences.com><BR>> Subject: [Histonet] Stripping
antibodies<BR>> To: <histonet@lists.utsouthwestern.edu><BR>>
Message-ID: <002401cc94ce$2619ca60$724d5f20$@com><BR>> Content-Type:
text/plain;
charset="us-ascii"<BR>><BR>> Hi!<BR>><BR>><BR>><BR>> Does
anyone have experience in IHC stripping antibodies from a tissue<BR>>
section and re-probing with different antibodies? What is the best way
to<BR>> do that? If you could share your stripping protocol or
experience I would<BR>> appreciate it -
thanks!<BR>><BR>><BR>><BR>>
Claire<BR>><BR>_______________________________________________<BR>Histonet
mailing list<BR>Histonet@lists.utsouthwestern.edu<BR><A
href="http://lists.utsouthwestern.edu/mailman/listinfo/histonet";>http://lists.utsouthwestern.edu/mailman/listinfo/histonet</A><BR></DIV></BLOCKQUOTE>
<DIV> </DIV> </DIV>
<DIV> </DIV><SPAN>On 29/10/11, <B>Amos Brooks
</B><amosbro...@gmail.com> wrote:</SPAN>
<BLOCKQUOTE style="BORDER-LEFT: #00f 1px solid; PADDING-LEFT: 13px;
MARGIN-LEFT: 0px">
<DIV>Hi,<BR> Stripping sites are usually found in the more
seedy areas of large<BR>cities ... Oh wait you meant ... nevermind :-) If you
developed the reaction<BR>with DAB, you may be in a difficult position. DAB is
a really strong<BR>reaction. You might try a Mallory bleach to remove it. 0.3%
sulfuric acid in<BR>5% potassium premanganate for 5 min followed by 5% oxalic
acid until it is<BR>clear (usually a couple of minutes). Unfortunately, I am
not sure what<BR>effect this will have on the epitopes you are looking for, but
this is<BR>usually what is used to clean precipitated DAB from instruments it
should<BR>work on the tissue too.<BR> If you use another
chromogen such as AEC it is much easier (to many<BR>people's dismay) to remove
this end product. Using alkaline phosphatase will<BR>be easy to remove the
chromogens as well. It would also be very easy to do<BR>this with fluorescent
tags too. Just remove the coverslip and dip the slide<BR>in ethanol and it will
remove the colored end product. Then just start over<BR>for the new
antigen.<BR><BR>Good luck,<BR>Amos<BR><BR>On Fri, Oct 28, 2011 at 11:50 AM,
<histonet-requ...@lists.utsouthwestern.edu<BR>> wrote:<BR><BR>>
Message: 2<BR>> Date: Thu, 27 Oct 2011 10:30:40 -0700<BR>> From: "Claire
Weston" <cwes...@valasciences.com><BR>> Subject: [Histonet] Stripping
antibodies<BR>> To: <histonet@lists.utsouthwestern.edu><BR>>
Message-ID: <002401cc94ce$2619ca60$724d5f20$@com><BR>> Content-Type:
text/plain;
charset="us-ascii"<BR>><BR>> Hi!<BR>><BR>><BR>><BR>> Does
anyone have experience in IHC stripping antibodies from a tissue<BR>>
section and re-probing with different antibodies? What is the best way
to<BR>> do that? If you could share your stripping protocol or
experience I would<BR>> appreciate it -
thanks!<BR>><BR>><BR>><BR>>
Claire<BR>><BR>_______________________________________________<BR>Histonet
mailing list<BR>Histonet@lists.utsouthwestern.edu<BR><A
href="http://lists.utsouthwestern.edu/mailman/listinfo/histonet";>http://lists.utsouthwestern.edu/mailman/listinfo/histonet</A><BR></DIV></BLOCKQUOTE>
<DIV> </DIV></DIV>
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